Tissue microarray builder manual set

ABSTRACT

The tissue micro-array (TMA) building manual set, which includes a well-known SZK tissue core punch extractor and a TMA block mould ( 1 ) characterized as follows: the TMA block mould ( 1 ) consists of a lower ( 2 ) and an upper ( 3 ) part, the lower part ( 2 ) is equipped with mandrels ( 4 ) at right angles to the base unit arranged in the middle into lines and columns, the upper part ( 3 ) has a margin-containing ( 7 ) cleavage ( 6 ), which receives the mandrels ( 4 ) when the TMA block mould ( 1 ) is assembled, the margin ( 7 ) and size of the cleavage ( 6 ) fit to the shape and size of the paraffin block holder (PBK), and the lower ( 2 ) and upper ( 3 ) parts are connected to each other with directing nipples ( 5 ) and threaded arms ( 9 ) fixed horizontally.

The object of our invention is a tissue micro-array (TMA) preparingmanual apparatus set, which includes a tissue core punch extractor and aTMA block mould with lower and upper parts.

The tissue micro-array preparing manual apparatus set is suitable formaking TMA series, which enables the fast and accurate “home-made”preparation of TMA series well-fitted to the traditional, routinehistology laboratory conditions.

TMA technology can be applied for the determination of diagnosticmarkers, as well as, parameters on the aggressiveness and other featuresof malignant tumors.

TMA is very useful for retrospective studies, i.e. for the investigationof thousands of molecular biological markers in thousands ofhistological specimens stored in pathology laboratories.

Similar to the DNA-chip, which allows the analysis of the genomemutations and abnormal gene expressions, the tissue micro-arrays enablethe parallel processing of biological specimens. By using only one TMA,researchers have he opportunity for simultaneous comparative examinationof various molecular parameters (DNA, RNA molecular biologicaltechniques and immunohistochemistry/immunocytochemistry of antigens) inhistological specimens of several malignant tumor tissues.

Numerous monoclonal antibodies, which determine the genome and phenotypeof intact and pathological (mainly malignant) cells, and are specificfor novel antigen-determinants, are being developed and introduced fordiagnostic and therapeutic applications (immunomorphology). Diagnosticmeasures based on genetic methods, which can identify certain segmentsof DNA and/or RNA, are increasingly used (molecular biology). Severallaboratories are involved in evaluating their tissue reactivity andpotential applicability in the most prevalent tumors.

As a consequence, this diagnostic technique indeed seems to be widelyused.

In the diagnostic technique, histology specimens from different tumortissues re embedded in properly arranged sets into paraffin for thepreparation of TMA. The TMA block is cut giving the various tumortissues as point-like items in the histological light microscopicsections. Since each TMA block may render hundreds of parallel, 3-4 □mthick serial sections, hundreds of molecular parameters can be examinedon the same micro-array. Several tumor markers can be analyzed withinweeks, while this would take months per tissue specimen usingtraditional method.

According to our current knowledge, reactions made on standard-sizehistological sections (from frozen and/or fixed-embedded tissues) andcytological samples (smears, sediments, imprints, cytocentrifugates,etc.) are useful for the identification o molecular markers of tumorsand for the prediction of disease progression. At the same time, merelylimited number of samples can be examined due to technical and financialreasons, the evaluation is time-consuming, and comparative, as well as,reproducible examinations among different laboratories are difficult tocarry out. First of all, the lack of reproducibility is the disadvantageof the so-called “multi-tissue block” technique that contains parts fromvarious tissues/blocks in a simple histological block. Moreover, thedistribution of individual molecular markers within the malignant tissueis often quite heterogeneous and, thus, a minute tissue part does notproperly represent the biological features of the entire tumor cellpopulation.

Application of the technique reminiscent of or rather based on the“chip” technology, i.e. the tissue micro-arrays, enables theinvestigation of hundreds or thousands of tissue specimens.

The knowledge in the field of medical biotechnological basic research isbeing multiplied, and its employment confirms both diagnostic andtherapeutic measures. Nevertheless, the related applied research,research and development (R&D), as well as, clinical testing processesrequire years or even a decade and proportional financial means. Ourinvention, the TMA building manual set shortens the necessary testingphase in an accurate and cost-efficient way.

The information in the U.S. Pat. No. 4,820,504 represents a well-knownand widely used procedure, which serves for the preparation of a samplecontaining multiple tissue specimens. Its advantages include the simpleprocedure that can be applied in almost every laboratory equipped withroutine and research facilities, low costs, and no need for procurementof new devices. However, the disadvantage of the technique is that theconsecutive, side by side embedded tissue samples are randomly situated,not organized into lines and columns, which makes the evaluation ratherdifficult, the automatic (image analyzing) processing almost impossibleor complicated and slow; the size of the individual tissue samples aremerely roughly similar to each other. New instrument does not belong tothe description of the procedure.

Our procedure and the object of our invention aims at addressing thesedisadvantages.

The U.S. Pat. No. 5,002,377 is n instrument, which organizes tissuesamples into regular lines and columns, and so gives a multiple tissueblock. Its patent holder is the same as that of the previous instrumentwho is a well-known expert on this field, despite this technique has notbecome widely used. Its obvious advantage is that application of thisinstrument enables the preparation and embedding of regular, almostidentical size tissue samples. Nevertheless, its disadvantage lies inthat the individual tissue columns may be, though only slightly, shiftedduring block preparation. The largest disadvantages of the instrumentare: its incompatibility with the most frequently applied and best knownlaboratory instruments (devices and semi-fixed assets) and, on the otherhand, the need for three additional tools and the significant manualskills for their application. The first instrument includes multipleparallel cutting plates, which can cut out tissue columns from tissuesamples, but can be applied exclusively on larger specimens. The othertwo components make possible the embedding of cut tissue columns into asingle paraffin block.

The U.S. Pat. No. 6,103,518 is a table device giving useful tissuemicro-arrays. The procedure is based on the well-oriented technique ofthe recipient places of the paraffin block and the sampling from theselected sites of donor blocks (tissue cores). Besides itsaforementioned advantages, its high price, complicated mechanicalsettings, and the need for one-by-one preparation of recipient holes ofthe recipient block represent serious drawbacks in the daily practice.

Publication document number WO 01/51910 presents a simple paraffin blockconstruction procedure that can be used for the production of tissuemicro-array series in which a mould that consists of several elementshas been formed. On using the mould a basic body is moulded that hasregularly arranged openings into which the tissue samples are thenplaced. Following this the basic body containing the tissue samples isheated up to the softening temperature limit in the interest of thebasic body being able to combine with the liquid paraffin poured ontoone of the surfaces in the following phase of the procedure and so forma single unit with it. This liquid paraffin layer serves to assist thefixing of the paraffin block holder to the paraffin block containing thecylindrical holes and the tissue samples contained in them.

However, a significant disadvantage of this solution is that theparaffin block that contains the cylinders with the tissue samples inthem is subjected to a significant heat effect. Due to the heat effectand the re-softening of the paraffin the tissue cylinders move, whichcan cause problems when making the sections. Another disadvantage of thesubsequent heat effect is that the amount of heat required to soften theparaffin may damage the tissue sample cylinders, which may causeirreversible damage from the point of view of the immune staining, asthis results in a change to the protein-based antigens and this can leadto the sample being unusable.

Another significant disadvantage of the solution is that in the paraffinblock poured in two phases, due to the shear forces occurring whenproducing the section the paraffin block may come apart at the borderbetween the two layers made at two separate points in time, as a resultof which the paraffin block may separate from the block holder so makingit impossible to make the desired sections from the tissue cylinders.

Examples suitable for the production of paraffin blocks used for theproduction of tissue samples can also be found in patent specificationregistration number U.S. Pat. No. 6,383,801 and furthermore, inpublication documents numbers WO 01/98525, WO 99/44063, WO 01/22086 andWO 01/42796.

The objective of our invention is to eliminate the disadvantages of theabove mentioned methods and to design a tissue micro-array preparingmanual apparatus set that is simple, cheap, and compatible with allwidely used histological laboratory fixed and semi-fixed assets. Theprinciple of applying the manual apparatus is that the preparation ofthe recipient block is separated from the sampling; therefore, thesamples can be previously prepared and stored elsewhere, then put intothe recipient block. The manual apparatus requires small space, is easyto clean, there is basically no need for maintenance, each part isresistant to external physical and chemical impacts, and the recipientblock can be prepared in one step.

Our invention is based on the recognition that the tissue micro-arraypreparing manual apparatus set should be made from elements that fittogether in their size and can be applied along with the instrumentsused under the traditional routine histological laboratory conditions.This tissue micro-array preparing manual apparatus set was developed tocontain a tissue core punch extractor and a TMA block mould with lowerand upper parts, and the traditional, most frequently used paraffinblock holders fit well to this latter one. The tissue core punchextractor is a well-known technical solution, i.e. its function is basedon punching. The punching tip is made as a punching tube with aninternal diameter of 1 or 2 mm. This punching tube receives the tissuecylinder (core) taken from a given place of the sample (donor block),which can be manually pushed out from the punching tip by help of apiston-like needle probe pushed back by a coil spring inside theinstrument. The external diameter of the needle probe is comparable tothe internal diameter of the punching tip, but due to its size it canfreely slide within the punching tip.

The TMA block mould is made up of two main elements: the lower and upperparts. The lower part is equipped with 1 or 2 mm wide mandrels,identical with the diameter of the punching tube, organized into linesand columns. Since the lower and upper parts are assembled togetherduring the casting of the paraffin block and the paraffin is poured intothe hole formed by the upper part, a margin should have been preparedthere, which is identical in its size and shape with the size and shapeof the generally used paraffin block holder.

The formation of the margin creates the opportunity to fix the paraffinblock holder itself to the empty paraffin block in one step whenmoulding the paraffin block. This, later on, makes it unnecessary tocarry out the subsequent heat treatment, which is essential in the knownprocedures, and so overcomes the disadvantageous consequences presentedabove deriving from the heat treatment.

At the lower part at least two vertical directing nipple are built intothe base unit, which ensures the parallel and intact removal of theparaffin block when the mould manual apparatus is taken apart. The equalwithdrawal of the two elements at the opening of the lower and upperpart of the TMA block mould is ensured by at least two diagonallyarranged threaded arms.

The elimination of the above discussed disadvantages is ensured by atissue micro-array preparing manual apparatus set with the followingcharacteristics: a by itself already known tissue core punch extractorand a TMA block mould, which latter has a lower and upper part; thelower part is organized in the middle into lines and columns, set upwith mandrels at right angles to the base unit; the upper part isprepared with a margin-containing cleavage, which receives the mandrelswhen the TMA block mould is assembled together; the margin and size ofthe cleavage fit to the shape and size of the paraffin block holder; thelower and upper parts are connected to each other with directing nipplesand threaded arms fixed horizontally; the lower part of the TMA blockmould has at least two vertical directing nipples, which verticallydetachably join into the bore-holes of the upper part; in the upper partof the TMA block mould there are at least two diagonal transitbore-holes, which receive the threaded arms and the endings of thethreaded arms sit on the lower part when the TMA block mould isassembled together.

Our invention, the TMA building manual set is presented in detail bymeans of drawings.

FIG. 1. Axonometric delineation of the TMA block mould.

FIG. 2. Axonometric delineation of the TMA block mould and paraffinblock holder of the TMA building manual set.

FIG. 3. The finished paraffin block with the tissue core punchextractor.

The 1 TMA block mould shown on FIG. 1. is made up of two major elements,i.e. 2 lower part and 3 upper part. The 2 lower part has 4 mandrels with1 or 2 mm diameter arranged into lines and columns. The diameter of the4 mandrel is identical with the diameter of the 8 punching tube of the14 tissue core punch extractor. On the 2 lower part there are 5directing nipples at right angles to the lower part, which ensure theparallel and intact removal of the paraffin block when the 1 TMA blockmould is dismantled. The size of the 3 upper part is identical with thatof the 2 lower part and in the middle it has a 7 margin-containing 6cleavage, which receives the 4 mandrels. The 7 margin of the 6 cleavageis made to receive the commercially available 12 paraffin block holdershown on FIG. 2. On the 2 lower part there are 5 directing nipples,which join the 10 transit bore-holes on the 3 upper part. Moreover, theadditional 11 transit bore-holes on the 3 upper part receives the 9threaded arms, which ensure the even and parallel opening of the twoparts of the assembled instrument cast with paraffin block.

The equal withdrawal of the two elements at the opening of the 2 lowerand 3 upper part of the 1 TMA block mould is ensured by at least twodiagonally arranged 9 threaded arms. The 14 tissue core punch extractorends in a 8 punching tube with an internal diameter of 1 or 2 mm.

The well-known 14 tissue core punch extractor receives the tissuecylinder taken from a given place of the sample (donor block), which canbe manually pushed out from the 8 punching tube by help of a piston-likeneedle probe pushed back by a coil spring inside the instrument.

The external diameter of the needle probe is comparable to the internaldiameter of the 8 punching tube, but due to its size it can freely slidewithin the 8 punching tip.

On preparing the paraffin block, the assembled 1 TMA block mould isfilled with paraffin through the 6 cleavage. After the paraffin is setwhen cooled down and removed from the instrument, it gives a 13 paraffinblock (recipient block) containing 1 or 2 mm wide cylindrical holes withparallel longitudinal axes organized into regular lines and columns.These holes receive the tissue cores having the same diameter taken outfrom the donor blocks by the 14 tissue core punch extractor, givingfinally the TMA block.

As it can be seen from our description, our objective was accomplishedby the development of our invention. The TMA building manual set is asimple, cheap manual instrument, which is easy to handle and clean,environment-friendly, and enables the fast and accurate preparation ofTMA under the traditional, routine histological laboratory conditions.

A further advantage is that due to the unique formation of the manualset it is possible to produce the paraffin block with holder in one stepwithout any subsequent heat treatment, which does not only reduce thetime required for the production but also has a favourable effect on themechanical characteristics of the finished piece, that is also includingthe shearing strength.

1. The tissue micro-array (TMA) building manual set, which includes awell-known tissue core punch extractor (14) and a TMA block mould (1),the TMA block mould (1) has a lower part (2), where this lower part (2)has mandrels (4) at right angles to the base unit arranged in the middleinto lines and columns, characterized with the TMA block mould (1) issupplemented with an upper part (3) the upper part (3) has amargin-containing (7) cleavage (6), which receives the mandrels (4) whenthe TMA block mould (1) is assembled, the margin (7) and size of thecleavage (6) fit to the shape and size of the paraffin block holder(12), and the lower (2) and upper (3) parts are connected to each otherwith directing nipples (5) and threaded arms (9) fixed horizontally. 2.The manual apparatus set as in the claim point No. 1 is characterizedwith: the lower part (2) of the TMA block mould (1) has at least twovertical directing nipples (5), which join the bore-holes (10) of theupper part (3) and are in vertical direction freely movable.
 3. Themanual apparatus set as in the claim point No. 1 is characterized with:the upper part (3) of the TMA block mould (1) has at least two diagonaltransmit bore-holes (11), which receive the threaded arms (9) sit on thelower part (2) when the TMA block mould (1) is assembled together.